Assessment of Commercial Real-Time PCR Assays for Detection of Malaria Infection in a Non-Endemic Setting

ABSTRACT.

Malaria control and elimination require prompt diagnosis and accurate treatment. Conventional methods such as rapid diagnostic tests (RDTs) and microscopy lack the characteristics to detect low parasitemias, commonly found in asymptomatic parasitemias and/or submicroscopic malaria carriers. On the contrary, molecular methods have higher sensitivity and specificity. This study evaluated the performance of two commercial real-time polymerase chain reaction (PCR) assays, RealStar^® Malaria PCR (RealStar-genus) and RealStar Malaria Screen&Type PCR (RealStar-species), compared with the reference Nested Multiplex Malaria PCR, for the detection of the main five Plasmodium species affecting humans. A total of 121 samples were evaluated. Values of sensitivity (98.9% and 97.8%) and specificity (100% and 96.7%) of the RealStar-genus and the RealStar-species assays, respectively, were very good. The limit of detection (LoD) for the RealStar-genus assay showed a mean value of 0.28 parasites/µL with Plasmodium falciparum samples; while, the LoD of the RealStar-species assay ranged from 0.09 parasites/µL for P. vivax to two parasites/µL for P. ovale. The time to complete a diagnosis was established in 4 hours. Our findings showed a very good concordance of both assays compared with the reference method, with a very good analytical sensitivity. RealStar-species assay was able to correctly characterize double and triple infections. Therefore, these RealStar assays have shown to be useful tools in malaria diagnosis in non-endemic countries and even endemic countries, and for malaria control in general, detecting low parasitemias with sensitivity similar to the most sensitive methods as nested PCR, but with lower time to get the results.

INTRODUCTION

According to the World Health Organization (WHO), there were an estimated 229 million cases of malaria in 2019.¹ Within the WHO malaria elimination and prevention programs, a prompt diagnosis and treatment is the most effective way to prevent mild malaria cases evolving into severe disease and death.

The most commonly used methods to detect malaria are microscopy and rapid diagnostic tests (RDTs). Microscopy is considered the gold standard test for malaria diagnosis. Thick blood smears are better in detecting low-density infections and both thick and thin smears are used to identify the stage of the parasites, determine the species, and quantify the parasite density. However, diagnosis by thick blood smear needs higher qualification and high experience to do it correctly.^2,3 RDTs are easy to use, inexpensive, and rapid methods for malaria diagnosis. However, most of them detect specifically P. falciparum and, in general, the sensitivity with the other Plasmodium species is poor.^4,5 Also, recently, as a result of the expansion of strains that do not express the histidine-rich protein 2 they can provide false negative results in P. falciparum infections.^4,5

Although these methods are very useful in the diagnosis of malaria symptomatic cases, they are far away from the diagnostic methods needed in setting of elimination, eradication, and avoiding the reintroduction of malaria in endemic countries and also in non-endemic countries, which report low parasitemias and asymptomatic individuals in immigrants visiting friends and relatives.⁶

In endemic areas, continuous exposures to Plasmodium parasites lead to the development of partial immunity, which can prevent the development of complications and, subsequently, it can lead to the presence of asymptomatic parasitemia and/or submicroscopic malaria (SMM) carriers.⁷ These cases provide a reservoir of parasites, not usually detected by the traditional diagnostic methods, but contributing to the persistence of malaria transmission.⁸ Besides, these asymptomatic and SMM malaria cases are prevalent in the malaria endemic regions, becoming a serious cause of concern as efforts are increasing toward the elimination of malaria parasites.

Thus, there is a need to increase the surveillance of asymptomatic parasitemias and SMM cases in endemic and non-endemic areas, to estimate the real percentage of cases, and to take efficient measures to avoid malaria transmission, for which the use of more sensitive methods to detect the parasite is essential.⁹

Diagnostic methods based on genomic amplification have shown sensitivity and specificity superior to conventional malaria diagnostic methods,^10–12 being able to detect submicroscopic infections^6,9 and mixed infections.¹³ Mixed Plasmodium spp. infections can lead to severe complications among malaria patients, meaning an important burden of malaria infections that are often unrecognized or underestimated.^14–16 Diagnostic procedures identifying mixed infections are crucial for therapeutic decisions and effective patient management.¹⁷

One of the most used molecular method for malaria diagnosis is nested polymerase chain reaction (PCR).^18,19 However, this method requires two steps, which increases the risks of contamination and the time of performing.²⁰ New methods such as loop-mediated isothermal amplification (LAMP), appear to be very sensitive and with possibilities to be used in resource-limited settings.^21,22 These methods were first described in 2000 by Notomi et al.,²³ as methods that amplify DNA with high specificity, efficiency, and rapidity under isothermal conditions, with the employment of Bst DNA polymerase. During amplification reaction, high amounts of DNA and pyrophosphate ion by-product are produced,²⁴ allowing different ways of detection of amplified products, such as turbidity, fluorescence, or colorimetric dyes.^25,26 This flexibility in the way of detection besides the isothermal nature of the process, which does not require a thermocycler or highly specific devices, have supported LAMP method such as an useful tool to be performed in low resource settings for malaria detection.^26–28 In addition, some commercial LAMP methods for malaria diagnosis have been developed.^29–31 Nevertheless, contamination can be also present in LAMP methods³¹ and independent amplification processes are needed for species identification.

Real-time PCR employs fluorescent labels to enable continuous monitoring of the PCR product formation throughout the reaction. It has been promoted as an automated, quantitative, and closed system that reduces the risk of cross-contamination.³² The use of probes linked to distinguishable dyes enables the simultaneous detection of the five malaria human-infecting species and the internal control in the corresponding detector channels of the PCR instrument.

The aim of this study was to evaluate the performance of two commercial real-time PCRs, RealStar^® Malaria PCR and RealStar Malaria Screen&Type PCR (Altona Diagnostics, Germany)³³ for the detection of the five more common species of Plasmodium that infect humans, including P. knowlesi, in a non-endemic malaria area, comparing to the Nested Multiplex malaria PCR (NM-PCR) used as reference method in the Spanish Malaria Diagnosis Reference Laboratory.^19,34 RealStar Malaria PCR (RealStar-genus) consists of one assay targeting Plasmodium spp., whereas RealStar Malaria Screen&Type PCR (RealStar-species) consists of two independent assays, one of them targeting P. vivax and P. falciparum and the other one targeting P. ovale, P. malariae, and P. knowlesi. This evaluation includes the performance of the test in single- and mixed-malarial infections, the analytical sensitivity, and the operational characteristics of both assays.

MATERIALS AND METHODS

Samples and study design.

Samples used in this retrospective study were provided by the repository of the Malaria and Emerging Parasitic Diseases Laboratory (Spanish National Biobanks Registry N: C.0001392). They belonged to travelers or immigrants coming from endemic malaria areas who attended different Spanish hospitals with malaria or other tropical diseases suspicion and were sent to the Spanish Malaria Reference Center within a project for the detection of SMM cases approved by the Medical and Health Research Ethics Committee (CEIC) of the Hospital Universitario 12 de Octubre (No. CEtm: 18/021). The samples were anonymized before their inclusion into the sample collection. Plasmodium knowlesi samples were collected in Malaysia under a project approved by the Medical Research Sub-Committee of the Malaysian Ministry of Health (NMRR-13-1064-18189).

A total of 121 samples were evaluated retrospectively. Fifteen from each Plasmodium species, 16 mixed-malarial infections (11 P. falciparum + P. ovale, 4 P. falciparum + P. malariae, and 1 P. ovale + P. malariae) and 30 malaria-negative samples, 15 of them negative for Plasmodium spp. but positive for other blood parasites (eight Leishmania infantum, one Loa loa, four Trypanosoma cruzi, one Dirofilaria immitis, and one Mansonella perstans). Initial diagnosis for malaria samples was made using the NM-PCR used as reference method in the Spanish Malaria Diagnosis Reference Laboratory. All samples were tested by both RealStar assays and by the reference method. In case of discrepancies, the samples were repeated by both methods.

Samples processing.

Blood samples in ethylenediaminetetraacetic acid (EDTA) were conserved at 4°C for less than 6 months, except for the samples corresponding to P. knowlesi that were processed for the study within 16 months of storage.

DNA was extracted from 200 μL of whole blood collected in EDTA tubes conserved a 4°C, using the QIAamp DNA Mini Blood Kit (QIAGEN^®, Germany), according to the manufacturer’s instructions. Internal control was added into each specimen, acting as a control for nucleic acid extraction procedure and as PCR inhibition control.³⁵ DNA was eluted in 100 μL of distilled sterile water and conserved at 4°C until the moment of performing PCR assays.

RealStar Malaria PCR and RealStar Malaria Screen & Type PCR.

RealStar-genus and RealStar-species assays were performed according to the manufacturer’s instructions. PCR cycling was performed on a RotorGene 6000 Cycler 5 PLEX plus HRM (Corbett, Australia). Samples were considered negative when the fluorescent signal was under the threshold at cycle 40.

Nested Multiplex Malaria PCR.

NM-PCR was performed according to manufacturer’s and original authors’ recommendations.^19,34 The method involves two multiplex PCR amplifications that target the small subunit ribosomal DNA (rDNA) gene of Plasmodium. The first reaction amplifies Plasmodium spp. and an internal amplification control and the second reaction, which uses the amplified DNA in the first reaction, enables the identification of the infecting species of P. vivax, P. falciparum, P. ovale, P. malariae, and P. knowlesi by the corresponding size of the amplified fragments in the agarose gel electrophoresis.³⁴ The commercial presentation of the NM-PCR (BIOMALAR-2 Gel Form kit, Biotools B&M Laboratories, Spain) is in individualized tubes (tubes ready-to-use) with all the necessary components for the reaction except the DNA and the water to complete the reaction volume. In the first reaction, 10 µL of DNA from the sample is added to the tube and for the second reaction 2 µL from the amplified product of the first PCR is used.

Reaction conditions for first PCR reaction were denaturation at 94°C for 7 minutes, followed by 40 cycles at 94°C for 20 seconds, 58°C for 20 seconds, and 72°C for 30 seconds. The final cycle was followed by an extension time at 72°C for 10 minutes. The conditions for the second PCR reaction were an initial denaturation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 15 seconds, 53°C for 15 seconds, and 72°C for 20 seconds, finishing with an extension phase at 72°C for 10 minutes.

Analytical sensitivity.

The analytical sensitivity or limit of detection (LoD) was calculated in two samples in duplicate of each Plasmodium species, three in the case of P. ovale, and two in mixed infections, using 10-fold serially diluted positive samples. Initial parasitemia of samples was calculated by microscopy, for P. falciparum, or by qPCR, for the rest of species and mixed infections, plotting Ct values on a standard curve obtained from samples with known parasitemia. The LoD was considered as the lowest parasite concentration in which samples and its duplicates were positive.³⁶

The LoD of the RealStar-genus was calculated using two P. falciparum–positive samples, with an initial parasitemia of 70,000 parasites/µL and 1,439 parasites/µL, respectively.

The LoD of the RealStar-species was calculated for the five human malaria species studied using two samples of P. falciparum, P. vivax, P. malariae, and P. knowlesi, and three samples in the case of P. ovale. The initial parasitemia for each sample was 70,000 and 1,439 parasites/µL for P. falciparum; 8,752 and 27,538 parasites/µL for P. vivax; 2,344 and 461 parasites/µL for P. malariae; 3,500 and 200 parasites/µL for P. knowlesi; and 1,215, 200, and 5,788 parasites/µL for P. ovale.

LOD was calculated with RealStar-species assay for two natural mixed infections: a sample containing P. falciparum (730 parasites/µL) + P. ovale (2,335 parasites/µL) and a sample containing P. malariae (84.7 parasites/µL) + P. ovale (360 parasites/µL).

Operational characteristics.

The time of performing was calculated from the initial moment the sample begins to be processed for the nucleic acid extraction until obtaining the results. The costs per sample were considered, without including the costs of controls included in each run or the duplication of samples. The costs related to the staff were estimated in time of performing the techniques. These costs are limited to performing the technique in Spain; in other countries the costs of kits may be completely different, including between institutions, although the relative differences will be similar.

Statistics.

The sensitivity (S), specificity (E), positive and negative predictive values (PPV and NPV), and kappa coefficient (k), with their respective 95% confidence intervals, were calculated using the EPI Dat (3.1) software package (Xunta de Galicia, Spain).³⁷

RESULTS

Both RealStar assays showed no cross-reactivity cases with the common blood parasites other than Plasmodium spp., such as L. infantum, T. cruzi, Loa loa, M. perstans, and D. immitis.

All the 121 samples yielded identical results in both RealStar assays according to the positive and negative results (100% of concordance).

Comparing the results of the RealStar-genus assay with results obtained with the reference method (NM-PCR), 120 samples out of 121 were concordant (99.2%). In this case, to make the comparison at the same level, it has been considered that any sample that gave an amplification fragment corresponding to P. falciparum, P. vivax, P. malariae, P. ovale, or P. knowlesi for the NM-PCR was considered Plasmodium spp. positive, and if no fragment appears, it would be Plasmodium spp. negative. The discrepant sample corresponded to a sample characterized as negative by the real-time PCR assay but positive for P. ovale by the reference method.

In the case of the RealStar-species assay, 118 out of 121 samples had the same results as the reference method, including 14 out of 16 mixed infections (97.5% of concordance). The three discordant results corresponded to the negative P. ovale sample (the same discrepant sample with the RealStar-genus assay) and two mixed-malarial infections. The discordant mixed-malarial infections were characterized with the reference method as P. falciparum + P. ovale and P. falciparum + P. malariae infections, while, with the RealStar-species assay, they were characterized as a triple infection (P. falciparum + P. ovale + P. malariae) and a single infection (P. malariae), respectively (Table 1).

Table 1.

Comparison of the RealStar^® Malaria Screen&Type PCR and the reference method (NM-PCR) in natural mixed infections

		Reference method (NM-PCR)
		P. falciparum+	P. falciparum+	P. ovale+
		P. malariae	P. ovale	P. malariae
RealStar Malaria Screen&Type	P. falciparum + P. malariae	3
	P. falciparum + P. ovale		10
	P. ovale + P. malariae			1
	P. falciparum + P. ovale + P. malariae		1
	P. malariae	1

This high concordance of both assays is reflected in the high values of sensitivity and specificity of the RealStar-genus and the RealStar-species assays, compared with the reference method (Table 2).

Table 2.

Values of sensitivity, specificity, positive and negative predictive values, and kappa coefficient of both RealStar^® assays compared with the reference method

	RealStar Malaria PCR		RealStar Malaria Screen&Type PCR
	Value	95% CI	Value	95% CI
Sensitivity	98.90%	96.21–100	97.80%	94.24–100
Specificity	100%	98.33–100	96.67%	88.58–100
PPV	100%	99.44–100	98.89%	96.17–100
NPV	96.77%	88.94–100	93.55%	83.29–100
Kappa coefficient	0.98	0.94–1	0.93	0.86–1

CI = confidence interval; NPV = negative predictive value; PPV = positive predictive value. The calculations of sensitivity, specificity, PPV, NPV, and kappa coefficient with the kit RealStar Malaria Screen&Type PCR were obtained considering the discordant results of mixed-malarial infections as follows: the triple infection of P. falciparum + P. ovale + P. malariae was considered a false positive and the single infection P. malariae as a false negative.

The LoD for the RealStar-genus assay was calculated with two P. falciparum samples, showing a value of 0.5 and 0.07 parasites/µL (mean value: 0.28 parasites/µL) (Table 3).

Table 3.

LoD of single malarial infections calculated for the RealStar^® Malaria PCR and the RealStar Malaria Screen&Type PCR assays

Real-time PCR Kit	Species	LoD sample 1	LoD sample 2	LoD sample 3	Mean + StD
RealStar genus	P. falciparum	0.5 p/µL	0.07 p/µL	–	0.28 ± 0.09 p/µL
RealStar species	P. falciparum	0.70 p/µL	0.14 p/µL	–	0.42 ± 0.15 p/µL
	P. ovale	0.58 p/µL	2.00 p/µL	1.20 p/µL	1.60 ± 1.01 p/µL
	P. vivax	0.09 p/µL	0.27 p/µL	–	0.18 ± 0.01 p/µL
	P. malariae	0.23 p/µL	0.46 p/µL	–	0.34 ± 0.02 p/µL
	P. knowlesi	0.30 p/µL	0.20 p/µL	–	0.25 ± 0.005 p/µL

LoD = limit of detection; RealStar-genus = RealStar Malaria PCR; RealStar-species = RealStar Malaria Screen&Type PCR; p/µL = parasites/µL; StD = standard deviation.

The LoD of the RealStar-species assay was calculated for each species individually and two natural mixed-malarial infections. In single infections, the LoD ranged from 0.09 parasites/µL for P. vivax to two parasites/µL for P. ovale (Table 3). In mixed infection of P. malariae + P. ovale, the LoD for each species was 8.47 and 0.36 parasites/µL, respectively; and in the P. falciparum + P. ovale infection, the LoD was 0.73 and 2.3 parasites/µL, respectively.

Regarding the operational characteristics, the turnaround time to complete a diagnosis, from the moment the sample is received until the moment the results are provided to clinicians, was estimated in 4 hours for the RealStar-assays: 1 hour for the management of the samples and DNA purification, 30 minutes for the master mix preparation and PCR setup, 2 hours to perform the real-time PCR, and 30 minutes for the analysis of results. Meanwhile, for the reference method, the diagnosis turnaround time was established in around 6 hours and 15 minutes: 1 hour for the management of the samples and DNA purification, 15 minutes for the first PCR setup, since no master mix preparation is necessary as the tubes are ready to use; 2 hours to perform the first PCR, 15 minutes for the second PCR setup, 2 hours to perform the second PCR, 30 minutes to run the automated electrophoresis, and 15 minutes for the analysis of results.

The hands-on time was much less for the RealStar-assays, including only the time of preparation of the samples, the PCR setup and the time of analysis, which was estimated to be 1 hour. For the reference method, the time required to prepare a second PCR and the electrophoresis must be added, amounting to 2 hours approximately.

Concerning the costs per sample of each method, they varied from 15 to 30€ for the RealStar-species assay and from 10 to 20€ for the RealStar-genus assay, depending on several circumstances; and about 5€ for the gel format NM-PCR assay.

DISCUSSION

In this study, we evaluated the performance of two real-time PCR assays for malaria diagnosis: the RealStar Malaria PCR Kit 1.0, which detects Plasmodium spp., and the RealStar Malaria Screen&Type PCR kit 1.0, which can detect P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi.

Both real-time PCR did not show unspecific amplifications or cross-reactivity with the other blood parasitic infections studied (L. infantum, T. cruzi, Loa loa, M. perstans, and D. immitis). No inhibition problems were observed with any of the RealStar assays, despite using some samples conserved at 4°C for more than 1 year; which is different from the percentage of 6.9% of inhibition reported in a previous study with freeze-thawed blood samples with the RealStar-species kit.¹²

Both assays, in comparison to the reference method (NM-PCR), showed a very good concordance (99.17% with the RealStar-genus kit and 97.5% with the RealStar-species assay). One of the discrepant results was common in the performance of both kits, a non-detected P. ovale infection. Likewise, both RealStar assays showed positive results for all the P. knowlesi–positive samples tested, although, in this case, only the RealStar-species kit can specifically identify P. knowlesi, similar to other commercial kits such as GenoAmp^® Real-Time PCR Malaria (Mediven^®, Malaysia) or the Malaria QuantiFast^TM Multiplex PCR (QIAGEN, Germany) among others; but different from the FTD Malaria differentiation kit (Fast Track Diagnostics, Malta), which specifically targets P. falciparum, P. vivax, P. malariae, and P. ovale, but no P. knowlesi.³⁸ Plasmodium knowlesi infections have place mainly in Malaysia,³⁹ where the number of cases increased from 1,600 to over 4,000 between 2016 and 2018.¹ However, P. knowlesi has also been imported by travelers from other countries who travel to a P. knowlesi endemic forested areas.^40,41 Because of the common misdiagnosis of P. knowlesi infections with traditional methods, molecular methods able to detect it, such as real-time PCR, are essential.

In this study, the diagnostic values (sensitivity, specificity, predictive values, and kappa coefficient) were very high, with a kappa coefficient very close to one, which means that both methods have a near perfect agreement to the reference test.

On the other hand, a key aspect of a proper malaria diagnosis is the detection of mixed infections.¹⁴ They are frequently unrecognized and diagnosed as single infections, mainly with traditional methods as microscopy and especially when one of the Plasmodium species has a low level of parasitemia,¹⁶ although this can also happen when PCR methods are used.⁴² This misdiagnosis of mixed infections may involve inadequate treatment, leading to relapses,¹⁶ damaging the patient’s health and the control of the disease in endemic areas. For this reason, it is important to make a correct diagnosis of mixed infections, and molecular methods are essential. In our study, the RealStar-genus kit showed positive results in the 16 tested mixed infections, but it lacks the identification of each of the Plasmodium involved species. By contrast, the RealStar-species assay did detected 15 out of the 16 mixed infections (93.75%). Just one mixed P. falciparum + P. malariae infection was misdiagnosed, because of the lack of P. falciparum detection, possibly due to low density of parasites. In contrast, a double infection (P. falciparum + P. ovale) was characterized as a triple infection (P. malariae + P. falciparum + P. ovale) with this kit. This result was obtained in duplicate and P. malariae appeared in the 27-cycle threshold (Ct) of the PCR, which suggests that it was probably a false negative result of the reference method. The reason for both discrepancies could be due to a low density of parasites, especially in the first case. Nevertheless, in the second case with a P. malariae Ct value of 27, other causes such as mismatches at the primer region or, more likely, competition between primers to get their target⁴³ may be the explanation. Therefore, the RealStar Malaria Screen&Type PCR kit showed a high ability to detect mixed infections, superior to other assays,³² and it was also able to detect triple-species infection, which are infrequent and are not usually identified by microscopy.¹⁵

Both RealStar assays had a similar LoD for P. falciparum detection. Likewise, the means of the LoD for all the species with the RealStar-species kit were very similar, except for P. ovale, with a range from 0.58 to 2 parasites/µL. Because of the disparity between the first two P. ovale samples, it was decided to test the LoD in a third P. ovale sample. This lower analytical sensitivity with P. ovale is probably the reason for the false negative result obtained with both assays, a sample infected with P. ovale with a parasitemia below the LoD for this species. In any case, these results show an analytical sensitivity higher than other reported real-time PCR assays, with LoDs of 0.7 and 2.9 parasites/µL for P. falciparum, 4 parasite/µL for P. ovale, and 1.5 parasite/µL for P. vivax;²⁰ and similar to those obtained with nested PCR methods.¹⁸

It is difficult to draw conclusions related to the analytical sensitivity in mixed infections since only two samples were assessed. In both cases, one of the species showed worse LoD than the one obtained in single infections with the same assay, being a possible explanation of a competitive inhibition suffered by the least represented species during the PCR.⁴³

With regards to the operational characteristics, the time to obtain the results in both RealStar assays was much shorter than with the reference method. This is also reflected in staff performance time, around 1 hour more for the reference method. However, the cost of these commercial assays is higher than the price of the NM-PCR, especially in the case of the RealStar-species assay. For this reason, it is recommended to use the RealStar-genus assay as a screening method and, in those positive cases, use the RealStar-species assay to characterize the species or species involved in the infection.

Therefore, our results show that both RealStar assays performed very similar to the reference method, although there are some advantages of the real-time PCR over nested PCR assays: 1) only one PCR step is needed compared with two steps in the nested PCR; 2) it is performed in a closed system and post-PCR handling is not required, which decreases the risks of contaminations; 3) the time to get the results is lower; and 4) it is possible to quantify the parasite load.²⁰ However, the cost over nested PCR is higher, which makes it more difficult to establish this technique in low resource settings, although it would be especially useful in countries where malaria is not endemic, where there is often a lack of expertise in malaria diagnosis microscopy, offering an accurate malaria diagnosis.

In conclusion, Altona RealStar assays equaled the very high sensitivity of the nested multiplex PCR assay, but with a lower time to get the results. These RealStar assays were able to detect low parasitemias and, in the case of the RealStar-species assay, it was able to speciate Plasmodium species. Therefore, these kits are useful tools in malaria diagnosis both in endemic countries, where low parasitemia infections prevent from malaria control and elimination and are usually not diagnosed; and in non-endemic countries, where lots of malaria cases report low parasitemias in immigrants returning from visiting friends and relatives and where conventional methods are not sensitive enough to detect them.